ABSTRACT Supplemental oxygen (hyperoxia) with/without continuous nasal positive airway pressure (CPAP) to preterm infants is associated with airway hyperreactivity (AHR) proceeding to wheezing and asthma. Understanding mechanisms by which hyperoxia and CPAP induce sustained AHR represents our long-term goal, and an unmet clinical need. We propose the initial stretch imposed by CPAP on more compliant bronchial airways in premature infants, particularly with added hyperoxia, is contributory. AHR involves greater [Ca2+]i and contractility of airway smooth muscle (ASM), and remodeling mediated partly by ASM proliferation. Our published studies and preliminary work using human fetal airway cells and neonatal mouse models show moderate hyperoxia (50% O2) and mechanical stretch not only enhance ASM contractility and proliferation, but also bronchial epithelial arginase, raising the question of whether and how epithelial arginase and neonatal AHR are linked. We propose the novel and intriguing idea that arginase-derived polyamines (e.g. spermine) have downstream effects on a novel ASM target: extracellular Ca2+ sensing receptor (CaSR). Although well-known for regulating body Ca2+, there is limited information on CaSR in lung, and none in postnatal airways. Preliminary data in developing human airways show high expression of ASM CaSR that responds to extracellular Ca2+ ([Ca2+]o) or spermine, and enhances [Ca2+]I, contractility and proliferation. Hyperoxia and stretch each increase CaSR expression and function. In neonatal mice exposed to 50% O2 and/or CPAP which show sustained AHR, epithelial arginase and ASM CaSR are increased, while inhibitors of arginase (nor-NOHA) and CaSR (calcilytic NPS2143) blunt AHR. Our overall hypothesis is that the epithelial arginase-ASM CaSR axis contributes to AHR in the context of hyperoxia and CPAP exposure in prematurity. We will examine this idea via 3 Aims. Aim 1: In developing bronchial epithelium, determine the effect of hyperoxia and/or stretch on the arginase pathway; Aim 2: In developing ASM, determine the role of CaSR in enhanced [Ca2+]i/contractility and proliferation induced by hyperoxia and/or stretch; Aim 3: In neonatal mouse models of hyperoxia and/or CPAP exposure, determine the role of the arginase-CaSR axis in AHR and airway remodeling. 18-22 week human fetal bronchial epithelial cells (fBECs) and ASM cells (fASM) are exposed to hyperoxia (<60% O2) +/- 0-15% stretch (representing 0 to high CPAP) with continuous 5% oscillations, mimicking clinical hyperoxia +/- CPAP in spontaneously breathing premies. fBEC arginase pathway and downstream effects (Aim 1), and role of fASM CaSR in [Ca2+]i/contractility and proliferation following hyperoxia +/- stretch (Aim 2) are examined, with signaling pathways such as RhoA/Rho kinase and MAPKs. In Aim 3, airway structural, functional and molecular changes are assessed in neonatal WT and smooth muscle CaSR KO mice exposed to 50% O2 +/- intermittent CPAP (3, 6, 9 cmH2O) for 7 days, with 14 days recovery (mimicking human conditions). Effects of inhibiting arginase (nor-NOHA) vs. CaSR (NPS2143) are tested towards establishing clinical significance.